Craniosynostosis is a significant health problem, occurring in more than 1/2500 live births. Current surgical treatments are imperfect, and a better understanding of the molecular, genetic, and cellular basis of cranial suture formation would lead to improved treatment strategies. Our understanding of the genetic regulation of suture formation has come in large part from identification of human mutations leading either to craniosynostosis or other defects in suture formation. In particular, the fact that haploinsufficiency for TWIST1 leads to craniosynostosis, and for the transcription factor RUNX2 to delayed and incomplete suture closure, demonstrates that the processes of skull and suture formation are sensitive to dosage of genes regulating osteoblast differentiation. The zebrafish has proven a valuable model system for the study of skeletogenesis, with substantial parallels to the processes in mammals. Our proposed experiments will advance the use of zebrafish for the study of skull bone and suture formation through three Specific Aims. First, we will analyze the normal process of cranial vault growth by Alizarin Red bone staining, and generate a quantitative morphometric description of normal skull growth. We will also characterize skull and suture growth at a molecular level, through analysis of gene expression patterns and the use of transgenic lines that express fluorescent marker genes in osteoblasts at different stages of differentiation. In the second aim, we will test the hypothesis that a transient population of neural crest cells play an important role in patterning the sutures by determining the dynamic contribution of neural crest to the sutures and skull bones, using genetic labeling to indelibly mark neural crest cells and their descendents. Finally, we will use the information obtained in the first two aims to guide us in further characterizing a mutant in the zebrafish osterix gene, which displays striking defects in skull and suture formation. We aim eventually to identify additional zebrafish mutants through forward genetics;our characterization of the normal processes of skull and suture formation will provide the necessary foundation of knowledge to determine the basis of the mutant phenotypes. Our work will also increase the utility of zebrafish as a powerful model system to contribute to our understanding of craniosynostosis and other defects in later skull development, and yield greater insights into the cellular and molecular processes of skull and suture formation shared among vertebrates. PUBLIC HEALTH RELEVANCE: Craniosynostosis and other defects in formation of cranial sutures represent a significant health problem, and current surgical treatments are imperfect. To better understand the processes leading to abnormal suture development, we will be characterizing the zebrafish as a novel genetic model system for skull bone and suture formation.